Studies On Separation, Characterization And Immune Activities Of Polysaccharides From Cordyceps Militaris Mycelium

Cordyceps militaris belongs to Cordyceps sinensis’ s different species of the same genus, which is the model species of Cordyceps. It is a kind of precious edible-medicinal fungi widely distributed around the world. Cordyceps militaris has a widely range of physiological activities and unique medicinal values. As one of the most important active ingredients in Cordyceps militaris, polysaccharides have become the focus of scientific research. At present, there have been a large number of studies on polysaccharides at home and abroad. However, little attention has been devoted to the study on the extraction of polysaccharides from Cordyceps militaris by subcritical water.Therefore, the aim of this research project was to isolate polysaccharides from Cordyceps militaris mycelium by subcritical water and to determine the optimal condition for extraction. Various polysacharide fractions which were isolated from crude polysaccharides were preliminarily analyzed in chemical structure, molecular chain conformation in aqueous solution and immune activity. And hope to provide theoretical basis for new polysaccharide drugs. The main results were as follows:(1) Response surface methodology based on single-factor experiments were applied to the optimization of the subcritical water extraction parameters of polysaccharides, The results showed that the yield of polysaccharide was 7.13% at p H 8,180℃ for 13 min with the liquid-solid ratio of 21:1(m L/g). Under the optimal conditions, actual yield was 7.13% and the theoretical yield was 7.71%. Compared with the traditional hot water extraction, subcritical water extraction method has obvious advantage in extraction time, extraction ratio and the yield was increased by 5.41%.(2) At the concentration of 0.6- 1.5 mg/m L, the reducing power of crude polysaccharides obtained by two extraction method gradually increase. And they had strong DPPH· eliminating ability in the range of 0.2 to 1 mg/m L,which were enhanced with the increase of concentration. In the same concentration, the DPPH· eliminating ability of polysaccharides extracted by subcritical water were stronger than that of hot water extraction, which the IC50 values were 0.314 and 0.324 mg/m L, respectively.(3) The Cordyceps militaris crude polysaccharides using subcritical water were fractionated by DEAE-52 cellulose column, Sephadex G-100 to obtained two homogeneous fraction CMP-W1 and CMP-S1, which showed white flocculent solid and faint yellow flocculent solid with the total sugar content 92.72% and 89.24%,respectively.(4) The weight-average molecular weight(Mw) of Cordyceps militaris polysaccharide CMP-W1 was determined by HPSEC-RI-MALLS to be about 366.2k Da and was composed of D-mannose, D-glucose, D-galactose with a relative molar ratio of 2.84:1:1.29. The main chain of CMP-W1 was formed of mannose and the branched chain contained galactose. Congo red reaction revealed that CMP-W1 had the triple-helical structure in aqueous solution. The particle size analysis indicated that the mean effective diameter of CMP-W1 was 992 nm with the dispersion 0.304. The chain conformation of CMP-W1 was observed by atomic force microscopy showing aggregation conformation which was intertwined by intermolecular or intramolecular interaction.(5) The weight-average molecular weight(Mw) of CMP-S1 was 460.3 k Da and was composed of D-mannose, D-glucose, D-galactose with a relative molar ratio of2.05:1:1.09. The main chain of CMP-S1 constituted mannose and glucose and the galactose was formed of branched chain. Congo red reaction revealed that CMP-S1 had not exist the triple-helical structure in aqueous solution. The particle size analysis indicated that the mean effective diameter of CMP-W1 was 713.9 nm and the dispersion was 0.257. The atomic force microscopy analysis showed that CMP-S1 existed as aggregation conformation in aqueous solution.(6) The results of cell culture showed that CMP-W1 and CMP-S1 can significantly promote the mice spleen lymphocytes proliferation and enhanced the immune function of mice by promoting the increase of Thl/Th2 type cytokines secretion in mouse spleen lymphocytes.